Detection of nucleic acids and proteins

ABSTRACT

The invention generally relates to methods for detecting a target nucleic acid and a target protein in a single assay.

RELATED APPLICATION

The present application is continuation of U.S. nonprovisional patentapplication Ser. No. 13/161,074, filed on Jun. 15, 2011, which is acontinuation-in-part of U.S. nonprovisional patent application Ser. No.12/034,698, filed Feb. 21, 2008, which claims the benefit of andpriority to U.S. provisional patent application Ser. No. 60/972,507,filed Sep. 14, 2007, the content of each of which is incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

The invention generally relates to methods for detecting a targetnucleic acid and a target protein in a single assay.

BACKGROUND

Diagnostic assays based upon multiple biomarkers have been used on onlya limited basis. For example, assays have been proposed in which geneexpression is measured in several genes in order to assess clinicalstatus. Also, multiple protein analytes have been used to screen for thepresence of any of multiple disorders when diagnosis is unclear.Generally, algorithms are used in order to assess the results of anystandard assay and, in particular to assess whether additional testingis needed. However, since different biomarker types provide differentinformative results, most assays have been limited to a single marker oranalyte per condition to be screened.

It is common to screen multiple analytes from the same sample fordifferent clinical indications. This is especially true when a patientpresents with ambiguous symptoms. For example, a single blood sample maybe screened for hematocrit, hepatitis antigen, HIV, and SARS. Each ofthose screens, however, is directed to a different clinical conditionand is tied into a different algorithm to produce separate results foreach of the clinical indications that the marker is intended to measure.Such a broad screen is used to rule out or rule in one or morediagnostic pathways in a situation in which diagnosis is ambiguous ordifficult.

Increasing the number of biomarkers in any screening assay increases theaccuracy of diagnosis. However, there is no assay that allows thescreening and/or diagnosis of a condition based upon a plurality ofbiomarkers. Therefore, there is a need in the art for assays anddiagnostic algorithms that allow screening and diagnosis of a conditionbased upon multiple biomarkers.

SUMMARY

The invention provides methods for assessing the clinical status of apatient. In particular, the invention provides methods for creating adiagnostic readout based upon analysis of multiple analytes orbiomarkers. In practice, methods of the invention provide the ability toscreen patients based upon a plurality of biomarkers in a single assayformat.

Methods of the invention are particularly useful in complex diagnosticassessment. The invention allows multiplex analysis of a plurality ofbiomarkers in order to increase the diagnostic power and accuracy of theresult. According to one aspect of the invention, a plurality ofdifferent biomarkers obtained from a patient sample are assessed. Theresults are then normalized and a diagnostic score is produced basedupon the normalized biomarker data. In a preferred embodiment, levels ofeach of a plurality of biomarkers in a patient sample are obtained. Eachbiomarker is then assigned a binary result (e.g., a 1 or a 0) based uponwhether the detected level of the biomarker in the patient sampleexceeds a predetermined threshold. Then, a cumulative score is obtainedby adding the binary results in order to produce a diagnostic score thatis used in clinical evaluation. In another preferred embodiment,biomarker results are weighted based upon known diagnostic criteriaand/or patient history, lifestyle, symptoms, and the like. The resultingaggregate weighted score is used for clinical assessment.

In certain embodiments of the invention, the readout of the plurality ofbiomarkers need not be binary. Rather, the readout may take intoconsideration the predictive value of each of the biomarkers for thecondition being assessed. This is a form of weighting based upon knownrisk factors, diagnostic criteria, and patient history and can be tunedto reflect the degree of confidence that one expects from the assay.Methods of the invention allow the generation of a signature based uponresults obtained from a plurality of biomarkers, wherein the signatureis indicative of the presence/absence of disease, the stage of disease,or prognostic factors (such as likelihood of recurrence, assessment ofresponse to treatment, and risk of developing disease).

Methods of the invention make use of the measurement of numerousdifferent markers that have a predictive relationship or possiblepredictive value in diagnosis, prognosis, therapeutic selection,therapeutic efficacy, physiological trait, and/or the likelihood ofrecurrence. The predictive power of multiplex diagnostic assessmentcreates a significant advantage in terms of both the specificity andsensitivity of the assay. The predictive power of the assay resides inits ability to take results from a number of different markers andcombine them into a single diagnostic signature or result thatencompasses the predictive power of each of the individual markers inorder to produce a highly-sensitive, highly-specific result.

Accordingly, in one embodiment of the invention, a plurality ofbiomarkers are measured in a sample obtained from a patient. Theplurality of biomarkers are selected from proteins (includingantibodies, enzymes, etc.), nucleic acids, carbohydrates, sugars,bacteria, viruses, pH, acids, bases, vitamins, ions, hormones, anddrugs. In some cases, for example in the case of nucleic acids andproteins, expression levels may be measured over time. In other cases,levels of a biomarker are obtained in whatever units may be appropriatefor that biomarker. Levels can optionally then be normalized across anentire panel of biomarkers or can be assigned a binary result based uponwhether a threshold is exceeded or not.

In some embodiments, results of a panel of biomarkers are used indiagnostic screening as they are obtained from an individual assay ofthe various biomarkers. In other cases, normalization occurs prior todiagnostic determination, and in still other cases, biomarker resultsare simply assigned a binary unit (e.g., a 1 or a 0). Cumulative resultsare then assessed based upon cumulative binary input (i.e., the sum ofall 1s and 0s) or on the basis of weighted averages or on the basis of asignature generated by the panel of markers chosen.

Markers chosen for multiplex diagnostic assays of the invention arechosen based upon their predictive value or suspected predictive valuefor the condition or conditions being diagnosed. Particular markers areselected based upon various diagnostic criteria, such as suspectedassociation with disease. The number of markers chosen is at thediscretion of the user and depends upon the cumulative predictiveability of the markers and the specificity/sensitivity of individualmarkers in the panel. A panel of markers can be chosen to increase theeffectiveness of diagnosis, prognosis, treatment response, and/orrecurrence. In addition to general concerns around specificity andsensitivity, markers can also be chosen in consideration of thepatient's history and lifestyle. For example, other diseases that thepatient has, might have, or has had can effect the choice of the panelof biomarkers to be analyzed. Drugs that the patient has in his/hersystem may also affect panel selection.

The invention is applicable to diagnosis and monitoring of any disease,either in symptomatic or asymptomatic patient populations. For example,the invention can be used for diagnosis of infectious diseases,inherited diseases, and other conditions, such as disease or damagecaused by drug or alcohol abuse. The invention can also be applied toassess therapeutic efficacy, potential for disease recurrence or spread(e.g. metastisis).

The invention is especially useful in screening for cancer. Examples ofbiomarkers associated with cancer include matrix metalloproteinase(MMP), neutrophil gelatinase-associated lipocalin (NGAL), MMP/NGALcomplex, thymosin β4, thymosin β10 thymosin β15, collagen like gene(CLG) product, prohibitin, glutathione-S-transferase, beta-5-tubulin,ubiquitin, tropomyosin, Cyr61, cystatin B, chaperonin 10, and profilin.Examples of MMPs include, but are not limited to, MMP-2, MMP-9,MMP9/NGAL complex, MMP/TIMP complex, MMP/TIMP1 complex, ADAMTS-7 orADAM-12, among others. Also, the patient sample from which a biomarkeris obtained is immaterial to the functioning of the invention. Preferredsample sources include blood, serum, sputum, stool, saliva, urine,cerebral spinal fluid, breast nipple aspirate, and pus.

Methods of the invention can be used on patients known to have adisease, or can be used to screen healthy subjects on a periodic basis.A subject can be screened for one or more diseases simultaneously usingmethods of the invention. Screening can be done on a regular basis(e.g., weekly, monthly, annually, or other time interval); or as a onetime event. The outcome of the analysis may be used to alter thefrequency and/or type of screening, diagnostic and/or treatmentprotocols. Different conditions can be screened for at different timeintervals and as a function of different risk factors (e.g., age,weight, gender, history of smoking, family history, genetic risks,exposure to toxins and/or carcinogens etc., or a combination thereof).The particular screening regimen and choice of markers used inconnection with the invention are determined at the discretion of thephysician or technician.

Threshold values for any particular biomarker and associated disease aredetermined by reference to literature or standard of care criteria ormay be determined empirically. In a preferred embodiment of theinvention, thresholds for use in association with biomarker panels ofthe invention are based upon positive and negative predictive valuesassociated with threshold levels of the marker. In one example, markersare chosen that provide 100% negative predictive value, in other wordspatients having values of a sufficient number of markers (which may beonly one) below assigned threshold values are not expected to have thedisease for which the screen is being conducted and can unambiguously bedetermined not to need further intervention at that time. Conversely,threshold values can be set so as to achieve approximately 100% positivepredictive value. In that case, a critical number of biomarker levelsabove that threshold are unambiguously associated with the need forfurther intervention. As will be apparent to the skilled artisan, forcertain biomarkers positive and negative predictive values do not haveto be 100%, but can be something less than that depending upon otherfactors, such as the patients genetic history or predisposition, overallhealth, the presence or absence of other markers for diseases, etc.

Further aspects and features of the invention will be apparent uponinspection of the following detailed description thereof.

DETAILED DESCRIPTION

The invention provides methods for clinical assessment in which a panelof different biomarkers obtained from a patient tissue or body fluidsample are analyzed and aggregated to produce a clinically-informativeresult. The result of using methods of the invention is increaseddiagnostic range and power.

According to the invention, multiple biomarkers are obtained from apatient sample (e.g., tissue or body fluid samples). Levels of thevarious markers are appropriately determined and a cumulativediagnostic/prognostic result is produced. Any number of differentbiomarkers can be chosen based upon the condition or conditions beingscreened. In many instances as, for example, in cancer, nucleic acidmutations, expression levels, methylation patterns and the like arescreened in coordination with protein levels. In an alternative example,steroid or protein hormones can be screened in conjunction with othertypes of markers and an aggregate diagnostic “score” can be produced.Other combinations of markers are apparent to those of ordinary skill inthe art and will depend upon the disease or condition for whichscreening is being conducted.

The invention allows the use of different analytes or biomarkers in asingle diagnostic algorithm in order to increase predictive power.According to the invention, multiple analytes are measured and themeasured outputs are converted into a single readout score or asignature that is predictive of clinical outcome. The readout can bebinary (e.g., 1/0, yes/no) or can be a point on a continuum thatrepresents a degree of risk of disease or severity or likely outcome(e.g., of treatment, recurrence, etc.). In any of these cases, thereadout is correlated to predictive outcomes at a desired level ofconfidence. For example, upon analysis of multiple analytes, a signaturecan be generated based upon the pattern of results obtained for theselected panel. That signature is then correlated to clinical outcomebased upon comparison to a training set with the same panel orempirically based upon prior results. The determination of individualanalyte results can also be placed into a bar code format that can bestructured to correlate with clinical outcome. Individual assay resultscan either be weighted or not and can either be normalized or notdepending upon the needs of the overall result.

By way of example, one aspect the invention provides a binary algorithmin which DNA and protein measurements are made in order to provide adiagnostic readout. In this example, an assay is conducted to determinewhether a mutation exists in a genomic region known to associate withcancer. For example, a single nucleotide polymorphism known to bepredictive of disease onset is first determined. There are numerousmeans for doing this, such as single base extension assays (e.g., U.S.Pat. No. 6,566,101, incorporated by reference herein). A resultindicating whether the mutation is present or not (1 or 0) is obtained.Several other DNA mutations can be measured as well and similarlyassigned a binary score for disease association. As many mutation-basedassays as are desired can be performed. The level of a protein orproteins known to be informative for cancer is also measured. This couldbe, for example, the tumor suppressor p53. It is determined whether thelevel of that protein exceeds a threshold amount known to be indicativeof the presence of disease. A binary result is also assigned to thisanalyte (e.g., 1 if threshold is exceeded and 0 if it is not). Finally,a quantitative RNA assay is performed to determine the level or levelsof diagnostically-relevant RNA expressed in the sample. A binary resultis obtained based upon the expression levels obtained for each RNAspecies measured, and comparison to known disease-associated thresholds.The result of all these assays is a series of binary outcomes that forma barcode-type readout that is assigned clinical status based upon apriori determinations of disease association for the entire markerpanel.

In another aspect of the invention, each of the assayed biomarkersproduces a quantitative result that is also assigned a weighted valuebased upon how much of the analyte is present in the sample relative toa predetermined threshold for the marker. For each marker, a resultabove the cutoff is given a weighted positive score (in this case basedupon amount present in excess of the cutoff) and those below thethreshold are given a weighted negative score. The weighted scores arethen assessed to provide an overall diagnostic readout.

There are numerous methods for determining thresholds for use in theinvention, including reference to standard values in the literature orassociated standards of care. The precise thresholds chosen areimmaterial as long as they have the desired association with diagnosticoutput.

Similarly, the biomarker chosen is immaterial to the operation of theinvention as long as the marker is associated with the disease for whichscreening is being conducted. Some biomarkers that have been associatedwith disease include nucleic acid markers (including but not limited toK-ras, K-ras2, APC, DCC, TP53, PRC I, NUSAPI, CAPZ, PFKP, EVER1, FLT1,ESPL I, AKAP2, CDC45L, RAMP, SYNGR2, NDRG1, ZNF533, and hypermethylatednucleic acid), proteins and peptides, carbohydrates, sugars, glycans,lipids, hormones (e.g., antidiuretic hormone (ADH), Adrenocorticotrophichormone (ACTH), growth hormone (GH), follicle stimulating hormone (FSH),luteinizing hormone (LH), estrogen (estradiol, estrone, estriol),progesterone, testosterone, dihydrotestosterone (DHT), inhibin,somatotropin, dehydroepiandrostenedione (DHEA), somatostatin, glucagon,insulin, thyrotropin, thyroid stimulating hormone (TSH), thyroxin,parathyroid hormone, corticotropin, cortisol, corticosteron,aldosterone, epinephrine, norepinephrine, prolactin, vasopressin,oxytocin, melanocyte stimulating hormone (MSH)), growth factors (e.g.,granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophagecolony stimulating factor (GM-CSF), nerve growth factor (NGF),neurotrophins, platelet-derived growth factor (PDGF), erythropeitin(EPO), thrmobopoeitin (TPO), myostatin (GDF-8), growth differentiationfactor (GDF-9), basic fibroblast growth factor (bFGF or FGF2), acidicfibroblast growth factor, fibroblast growth factor receptor 3 (FGFR3),epidermal growth factor (EGF), hepatocyte growth factor (HGF), humanstem cell factor (SCF), tumor necrosis factor (TNF), tumor necrosisfactor-β (TNF-β), tumor necrosis factor-α (TNF-α), vascular endothelialgrowth factor (VEGF), transforming growth factor-β (TGF-β), transforminggrowth factor-α (TGF-α), insulin-like growth factor-I (IGF-II),insulin-like growth factor-II (IGF-II), and colony stimulating factor(CSF)), cytokines (e.g., IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IFN-α, IFN-β, and IFN-γ),proteins (e.g., Matrix metalloproteinases (MMPs) such as MMP2, MMP9,neutrophil gelatinase-associated lipocalin (NGAL), MMP/NGAL complex,thymosin β15, thymosin β16, collagen like gene (CLG) product,prohibitin, glutathione-S-transferase, beta-5-tubulin, ubiquitin,tropomyosin, Cyr61, cystatin B, chaperonin 10, profilin,Alpha-fetoprotein, Carcinoembryonic antigen, Epidermal growth factorreceptor, Kallikrein 3 (prostate specific antigen), Vascular endothelialgrowth factor A, VEGF, Albumin, CA 125, Calcitonin, Chromogranin A(parathyroid secretory protein 1), Corticotropin-lipotropin containsACTH, Estrogen receptor 1, Gastrin, Progesterone receptor, Prolactin,5100 alpha chain, Somatostatin, Thyroglobulin, V-erb-b2, Her2/neu,Antigen identified by monoclonal antibody Ki-67, B-cell CLUlymphoma 2,BCL2-associated X protein, Beta-2-microglobulin, Breast cancer 1 earlyonset, BRCA1, CA 15.3, CA 19.9, Cadherin 1 type 1 E-cadherin(epithelial), Caspase 3, CD44 antigen, Cellular tumor antigen p53,Coagulation factor II, prothrombin, Colony stimulating factor 2(granulocyte-macrophage), Colony stimulating factor 3 (granulocyte),C-reactive protein, Cyclin D1, Cyclin-dependent kinase inhibitor 1, p21,Erythropoietin, Fibrinogen alpha/alpha-E chain, Follicle-stimulatinghormone, Gamma enolase, Insulin, Interferon gamma, Interleukin 2,Interleukin 6, k-ras, Neprilysin, CD10, Transferrin, Trypsin, Tumornecrosis factor (TNF-alpha), Tumor necrosis factor receptor superfamilymember 6, fas, Von Willebrand Factor, Chemokine, Chitinase-3 likeprotein 1, YKL-40, Choriogonadotropin beta chain, Colony stimulatingfactor 1 (macrophage), Haptoglobin-1, Hepatocyte growth factor, Inhibin,Interferon-alpha/beta receptor alpha chain, Interferon-alpha/betareceptor beta chain, Kallikrein 10, Kallikrein 11, Kallikrein 6, Matrixmetalloproteinase 3, ADAM-12, Small inducible cytokine A21 (CCL21)soluble IL-2R alpha, Somatotropin growth factor, growth hormone, Breastcancer 2 early onset, BRCA2, Catenin Beta 1, Cathepsin D, CD15, Desmin,DNA-(apurinic or apyrimidinic site) lyase, APEX, Lutropin beta chain,Luteinizing hormone, Parathyroid Hormone, Proliferating cell nuclearantigen, Tumor necrosis factor ligand superfamily member 8 (CD30ligand), V-myc myelocytomatosis viral oncogene homolog (avian), Tumornecrosis factor ligand superfamily member 8 (CD30),17beta-Hydroxysteroid dehydrogenase type 1 (17HSD1), Acid phosphataseprostate, Adrenomedullin, Aldolase A, bone-specific Alkalinephosphatase, Alkaline phosphatase, placental type, Alpha-1-acidglycoprotein 1, orosomucoid, Alpha-1-antitrypsin, alpha-2-HS-glycoprotein, Alpha-2-macroglobulin, Alpha-lactalbumin, Angiogeninribonuclease RNase A family 5, Angiopoietin 1, Angiopoietin 2,Antileukoproteinase 1, SLPI, Apolipoprotein A1, Apolipoprotein A-II,Apolipoprotein C-1, Apolipoprotein C-III, Bone sialoprotein II,Brain-derived neurotrophic factor, Breast cancer metastasis-suppressor1, CA 27.29, CA 72-4, Cathepsin B, CC chemokine 4, HCC-4, CD44 variantV5 soluble, Ceruloplasmin, Cervical cancer 1 protooncogene protein p40,Chemokine (C—C motif) ligand 4 Small inducible cytokine A4 (CCL4),MIP-1-beta, Claudin-3, Claudin-4, Clusterin, Coagulation factor III,Coagulation factor XIII A chain, Coagulation factor XIII B chain,Collagen I c-terminal telopeptide, Complement component 3, Complementcomponent 4, Complement component 7, Complement factor H relatedprotein, Cyclin-dependent kinase 6, Cyclooxygenase-2, Cystatin A,Cystatin B, Cystatin C, Cytokeratin 8, Diazepam binding inhibitor,Endoglin, Endothelin 1, Epidermal growth factor, E-selectin, Ferritin H,Fibroblast growth factor 2 (basic), Fibronectin 1, Flt-3 ligand,Fms-related tyrosine kinase 1, VEGFRI, Follistatin,Fructose-bisphosphate aldolase B, Fructose-bisphosphate aldolase C,Geminin, Glucose-6-phosphate isomerase, Glypican-3, n-terminal, Growtharrest and DNA-damage-inducible alpha, Immunosuppressive acidic protein,Insulin-like growth factor 1 (somatomedin C), Insulin-like growth factor2 (somatomedin A), Insulin-like growth factor binding protein 1,Insulin-like growth factor binding protein 2, Insulin-like growth factorbinding protein 3, Intercellular Adhesion Molecule 1, Interferon alpha1, Interleukin 1 alpha, Interleukin 1 beta, Interleukin 10, Interleukin12A, Interleukin 16, Interleukin 5, Interleukin 6 receptor, Interleukin6 signal transducer, Interleukin 7, Interleukin 8, Interleukin 9,Interleukin-1 receptor antagonist protein, IRAP, Kallikrein 14 (hK14),Kallikrein 2 prostatic, Kallikrein 5, Kallikrein 7, Kallikrein 8,Kallikrein 18, Kallikrein 8, Keratin 18, Keratin, type I cytoskeletal19, cytokeratin 19, Kit ligand, Lactotransferrin, Leptin, L-selectin,Luteinizing hormone-releasing hormone receptor, Mac-2 Binding Protein90K, Mammaglobin B, Mammary Serum, Antigen, Mast/stem cell growth factorreceptor, Melanoma-inhibiting activity, Membrane cofactor protein, CD46antigen, Mesothelin, Midkine, MK-1 protein, Ep-CAM, Myoblastdetermination protein 1, Nerve growth factor beta, Netrin-1,Neuroendocrine secretory protein-55, Neutrophil defensin 1, Neutrophildefensin 3, Nm23-H 1, OVX1, OX40, p65 oncofetal protein, Pancreaticsecretory trypsin inhibitor, TATI, Parathyroid hormone-related protein,Pcaf, P300/CBP-associated factor, Pepsinogen-1, Placental specifictissue protein 12 Plasma retinol-binding protein, Plasminogen (ContainsAngiostatin), Platelet endothelial cell adhesion molecule, PECAM-1,Platelet factor 4, Platelet-derived growth factor beta polypeptide,Platelet-derived growth factor receptor alpha polypeptide, Pregnancyzone protein, Pregnancy-associated plasma protein-A, Prostate secretoryprotein PSP94, P-selectin, PSP94 binding protein, Pyruvate kinase,isozymes M1/M2, Riboflavin carrier protein, 100 beta chain, Secretedphosphoprotein 1, osteopontin, Serine (or cysteine) proteinase inhibitorGlade B, maspin, Serine (or cysteine) proteinase inhibitor Glade E,PAI-1, Serum amyloid alpha-1, Serum paraoxonase/arylesterase 1, Smallinducible cytokine A14 CCL14, Small inducible cytokine A18(CCL18),MIP-4, Small inducible cytokine A2(CCL2), Small inducible cytokineA3(CCL3), Macrophage inflammatory protein 1-alpha, Small induciblecytokine B5(CXCL5), Squamous cell carcinoma antigen 1, Squamous cellcarcinoma antigen 2, Survivin, Syndecan-1, synuclein-gamma, TEK tyrosinekinase endothelial, Tie-2, Tenascin, Tetranectin, TGF-beta receptor typeIII, Thiredoxin reductase 1, Thrombopoietin, Thrombopoietin 1, Thymidinkinase, Tissue inhibitor of metalloproteinase1, Tissue inhibitor ofmetalloproteinase2, Tissue-type plasminogen activator, tPA, Transferrinreceptor (p90 CD71), Transforming growth factor alpha, Transforminggrowth factor beta 1, transthyretin, Tropomyosin 1 alpha chain(Alpha-tropomyosin), Tumor necrosis factor (ligand) superfamily member5, CD154, Tumor necrosis factor (ligand) superfamily member 6, Fasligand, Tumor necrosis factor ligand superfamily member 13B, TALL-1,Tumor necrosis factor receptor superfamily member 11 B, osteoprotegerin,Tumor necrosis factor receptor superfamily member 1A p60 TNF-RI p55CD120a, TNFR1, Tumor necrosis factor receptor superfamily member 1B,TNFR2, Urokinase plasminogen activator surface receptor, U-PAR, Vascularcell adhesion molecule 1, Vascular endothelial growth factor receptor 2,Vasoactive intestinal peptide, VEGF (165)b, Vitamin K dependent proteinC, Vitronectin, and X box binding protein-1), antibodies, or anycombination thereof.

In another aspect of the invention, a single assay is used to detectboth nucleic acids and proteins from a single sample. Biological samplesusually do not include a sufficient amount of DNA for detection. Acommon technique used to increase the amount of nucleic acid in a sampleis to perform PCR on the sample prior to performing an assay thatdetects the nucleic acids in the sample. PCR involves thermal cycling,consisting of cycles of repeated heating and cooling of a reaction forDNA melting and enzymatic replication of the DNA. Most PCR protocolsinvolve heating DNA to denature the double stranded DNA in the sample,cooling the DNA to allow for annealing of primers to the single-strandedDNA to form DNA/primer complexes and binding of a DNA polymerase to theDNA/primer complexes, and re-heating the sample so that the DNApolymerase synthesizes a new DNA strand complementary to thesingle-stranded DNA. This process amplifies the DNA in the sample andproduces an amount of DNA sufficient for detection by standard assaysknown in the art, such as Southern blots or sequencing.

A problem with detecting both nucleic acids and proteins in a singleassay is that the temperatures used for PCR adversely affect proteins inthe sample, making the proteins undetectable by methods known in theart, such as western blots. For example, the required heating step in aPCR reaction brings the sample to a temperature that can result inirreversible denaturation of proteins in the sample and/or precipitationof proteins from the sample. Additionally, thermal cycling, i.e.,repeated heating and cooling, can cause proteins in a sample to adopt anon-native tertiary structure. Once denatured, the proteins usuallycannot be detected by standard protein assays such as western blots,immunoprecipitation, or immunoelectrophoresis. Therefore a need existsfor a single assay that can analyze both proteins and nucleic acids in asample.

Methods of the present invention can detect a target nucleic acid and atarget protein in a single assay. In certain embodiments, methods of theinvention are accomplished by adding an aptamer to a sample that binds atarget protein in the sample to form an aptamer/protein complex. Anaptamer (nucleic acid ligand) is a nucleic acid macromolecule (e.g. DNAor RNA) that binds tightly to a specific molecular target, such as aprotein. Since an aptamer is composed of DNA or RNA, it can be PCRamplified and can be detected by standard nucleic acid assays. PCR maythen be used to amplify the nucleic acids and the aptamer in the sample.The amplified nucleic acids and aptamer may then be detected usingstandard techniques for detecting nucleic acids that are known in theart. In particular embodiments, the detection method is sequencing.Detection of the aptamer in the sample indicates the presence of thetarget protein in the sample.

As used herein, “aptamer” and “nucleic acid ligand” are usedinterchangeably to refer to a nucleic acid that has a specific bindingaffinity for a target molecule, such as a protein. Like all nucleicacids, a particular nucleic acid ligand may be described by a linearsequence of nucleotides (A, U, T, C and G), typically 15-40 nucleotideslong. Nucleic acid ligands can be engineered to encode for thecomplementary sequence of a target protein known to associate with thepresence or absence of a specific disease.

In solution, the chain of nucleotides form intramolecular interactionsthat fold the molecule into a complex three-dimensional shape. The shapeof the nucleic acid ligand allows it to bind tightly against the surfaceof its target molecule. In addition to exhibiting remarkablespecificity, nucleic acid ligands generally bind their targets with veryhigh affinity, e.g., the majority of anti-protein nucleic acid ligandshave equilibrium dissociation constants in the picomolar to lownanomolar range.

Aptamers used in the methods of the invention depend upon the targetprotein to be detected. Nucleic acid ligands for specific targetproteins may be discovered by any method known in the art. In oneembodiment, nucleic acid ligands are discovered using an in vitroselection process referred to as SELEX (Systematic Evolution of Ligandsby Exponential enrichment). See for example Gold et al. (U.S. Pat. Nos.5,270,163 and 5,475,096), the contents of each of which are hereinincorporated by reference in their entirety. SELEX is an iterativeprocess used to identify a nucleic acid ligand to a chosen moleculartarget from a large pool of nucleic acids. The process relies onstandard molecular biological techniques, using multiple rounds ofselection, partitioning, and amplification of nucleic acid ligands toresolve the nucleic acid ligands with the highest affinity for a targetmolecule. The SELEX method encompasses the identification ofhigh-affinity nucleic acid ligands containing modified nucleotidesconferring improved characteristics on the ligand, such as improved invivo stability or improved delivery characteristics. Examples of suchmodifications include chemical substitutions at the ribose and/orphosphate and/or base positions. There have been numerous improvementsto the basic SELEX method, any of which may be used to discover nucleicacid ligands for use in methods of the invention.

In methods of the invention, aptamers are introduced to the sample tobind the target protein. Certain of the aptamers bind the protein(s) ofinterest in the sample to form aptamer/protein complexes. The unboundaptamers are then separated and/or removed from sample using standardmethods known in the art. See for example, Schneider et al., U.S. PatentApplication Publication Number 2009/0042206, the content of which isincorporated by reference herein in its entirety.

Amplification refers to production of additional copies of a nucleicacid sequence. See for example, Dieffenbach and Dveksler, PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. (1995), thecontents of which is hereby incorporated by reference in its entirety.The amplification reaction may be any amplification reaction known inthe art that amplifies nucleic acid molecules, such as polymerase chainreaction, nested polymerase chain reaction, polymerase chainreaction-single strand conformation polymorphism, ligase chain reaction,strand displacement amplification and restriction fragments lengthpolymorphism.

In certain methods of the invention, the target nucleic acid and thenucleic acid ligand are PCR amplified. PCR refers to methods by K. B.Mullis (U.S. Pat. Nos. 4,683,195 and 4,683,202, hereby incorporated byreference) for increasing concentration of a segment of a targetsequence in a mixture of genomic DNA without cloning or purification.The process for amplifying the target nucleic acid sequence and nucleicacid ligand includes introducing an excess of oligonucleotide primersthat bind the nucleic acid and the nucleic acid ligand, followed by aprecise sequence of thermal cycling in the presence of a DNA polymerase.The primers are complementary to their respective strands of the targetnucleic acid and nucleic acid ligand.

To effect amplification, the mixture of primers are annealed to theircomplementary sequences within the target nucleic acid and nucleic acidligand. Following annealing, the primers are extended with a polymeraseso as to form a new pair of complementary strands. The steps ofdenaturation, primer annealing and polymerase extension can be repeatedmany times (i.e., denaturation, annealing, and extension constitute onecycle; there can be numerous cycles) to obtain a high concentration ofan amplified segment of a desired target and nucleic acid ligand. Thelength of the amplified segment of the desired target and nucleic acidligand is determined by relative positions of the primers with respectto each other, and therefore, this length is a controllable parameter.

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level that can be detected by severaldifferent methodologies (e.g., staining, hybridization with a labeledprobe, incorporation of biotinylated primers followed by avidin-enzymeconjugate detection, incorporation of 32P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment).

In one embodiment of the invention, the target nucleic acid and nucleicacid ligand can be detected using detectably labeled probes. Nucleicacid probe design and methods of synthesizing oligonucleotide probes areknown in the art. See, e.g., Sambrook et al., DNA microarray: AMolecular Cloning Manual, Cold Spring Harbor, N.Y., (2003) or Maniatis,et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y., (1982), the contents of each of which are herein incorporated byreference herein in their entirety. Sambrook et al., Molecular Cloning:A Laboratory Manual (2^(nd) Ed.), Vols. 1-3, Cold Spring HarborLaboratory, (1989) or F. Ausubel et al., Current Protocols In MolecularBiology, Greene Publishing and Wiley-Interscience, New York (1987), thecontents of each of which are herein incorporated by reference in theirentirety. Suitable methods for synthesizing oligonucleotide probes arealso described in Caruthers, Science, 230:281-285, (1985), the contentsof which are incorporated by reference.

Probes suitable for use in the present invention include those formedfrom nucleic acids, such as RNA and/or DNA, nucleic acid analogs, lockednucleic acids, modified nucleic acids, and chimeric probes of a mixedclass including a nucleic acid with another organic component such aspeptide nucleic acids. Probes can be single stranded or double stranded.Exemplary nucleotide analogs include phosphate esters of deoxyadenosine,deoxycytidine, deoxyguanosine, deoxythymidine, adenosine, cytidine,guanosine, and uridine. Other examples of non-natural nucleotidesinclude a xanthine or hypoxanthine; 5-bromouracil, 2-aminopurine,deoxyinosine, or methylated cytosine, such as 5-methylcytosine, andN4-methoxydeoxycytosine. Also included are bases of polynucleotidemimetics, such as methylated nucleic acids, e.g., 2′-O-methRNA, peptidenucleic acids, modified peptide nucleic acids, and any other structuralmoiety that can act substantially like a nucleotide or base, forexample, by exhibiting base-complementarity with one or more bases thatoccur in DNA or RNA.

The length of the nucleotide probe is not critical, as long as theprobes are capable of hybridizing to the target nucleic acid and nucleicacid ligand. In fact, probes may be of any length. For example, probesmay be as few as 5 nucleotides, or as much as 5000 nucleotides.Exemplary probes are 5-mers, 10-mers, 15-mers, 20-mers, 25-mers,50-mers, 100-mers, 200-mers, 500-mers, 1000-mers, 3000-mers, or5000-mers. Methods for determining an optimal probe length are known inthe art. See, e.g., Shuber, U.S. Pat. No. 5,888,778, hereby incorporatedby reference in its entirety.

Probes used for detection may include a detectable label, such as aradiolabel, fluorescent label, or enzymatic label. See for exampleLancaster et al., U.S. Pat. No. 5,869,717, hereby incorporated byreference. In certain embodiments, the probe is fluorescently labeled.Fluorescently labeled nucleotides may be produced by various techniques,such as those described in Kambara et al., Bio/Technol., 6:816-21,(1988); Smith et al., Nucl. Acid Res., 13:2399-2412, (1985); and Smithet al., Nature, 321: 674-679, (1986), the contents of each of which areherein incorporated by reference in their entirety. The fluorescent dyemay be linked to the deoxyribose by a linker arm that is easily cleavedby chemical or enzymatic means. There are numerous linkers and methodsfor attaching labels to nucleotides, as shown in Oligonucleotides andAnalogues: A Practical Approach, IRL Press, Oxford, (1991); Zuckerman etal., Polynucleotides Res., 15: 5305-5321, (1987); Sharma et al.,Polynucleotides Res., 19:3019, (1991); Giusti et al., PCR Methods andApplications, 2:223-227, (1993); Fung et al. (U.S. Pat. No. 4,757,141);Stabinsky (U.S. Pat. No. 4,739,044); Agrawal et al., TetrahedronLetters, 31:1543-1546, (1990); Sproat et al., Polynucleotides Res.,15:4837, (1987); and Nelson et al., Polynucleotides Res., 17:7187-7194,(1989), the contents of each of which are herein incorporated byreference in their entirety. Extensive guidance exists in the literaturefor derivatizing fluorophore and quencher molecules for covalentattachment via common reactive groups that may be added to a nucleotide.Many linking moieties and methods for attaching fluorophore moieties tonucleotides also exist, as described in Oligonucleotides and Analogues,supra; Guisti et al., supra; Agrawal et al, supra; and Sproat et al.,supra

The detectable label attached to the probe may be directly or indirectlydetectable. In certain embodiments, the exact label may be selectedbased, at least in part, on the particular type of detection methodused. Exemplary detection methods include radioactive detection, opticalabsorbance detection, e.g., UV-visible absorbance detection, opticalemission detection, e.g., fluorescence; phosphorescence orchemiluminescence; Raman scattering. Preferred labels includeoptically-detectable labels, such as fluorescent labels. Examples offluorescent labels include, but are not limited to,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl)]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; alexa;fluorescien; conjugated multi-dyes; Brilliant Yellow; coumarin andderivatives; coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives; eosin, eosin isothiocyanate, erythrosin and derivatives;erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives; 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™. Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives; Atto dyes, Cy3; Cy5; Cy5.5; Cy7; IRD 700;IRD 800; La Jolta Blue; phthalo cyanine; and naphthalo cyanine. Labelsother than fluorescent labels are contemplated by the invention,including other optically-detectable labels.

Detection of a bound probe may be measured using any of a variety oftechniques dependent upon the label used, such as those known to one ofskill in the art. Exemplary detection methods include radioactivedetection, optical absorbance detection, e.g., UV-visible absorbancedetection, optical emission detection, e.g., fluorescence orchemiluminescence. Devices capable of sensing fluorescence from a singlemolecule include scanning tunneling microscope (siM) and the atomicforce microscope (AFM). Hybridization patterns may also be scanned usinga CCD camera (e.g., Model TE/CCD512SF, Princeton Instruments, Trenton,N.J.) with suitable optics (Ploem, in Fluorescent and Luminescent Probesfor Biological Activity Mason, T. G. Ed., Academic Press, Landon, pp.1-11 (1993)), such as described in Yershov et al., Proc. Natl. Acad.Sci. 93:4913 (1996), or may be imaged by TV monitoring. For radioactivesignals, a phosphorimager device can be used (Johnston et al.,Electrophoresis, 13:566, 1990; Drmanac et al., Electrophoresis, 13:566,1992; 1993). Other commercial suppliers of imaging instruments includeGeneral Scanning Inc., (Watertown, Mass. on the World Wide Web atgenscan.com), Genix Technologies (Waterloo, Ontario, Canada; on theWorld Wide Web at confocal.com), and Applied Precision Inc.

In certain embodiments, the target nucleic acid or nucleic acid ligandor both are quantified using methods known in the art. A preferredmethod for quantitation is quantitative polymerase chain reaction(QPCR). As used herein, “QPCR” refers to a PCR reaction performed insuch a way and under such controlled conditions that the results of theassay are quantitative, that is, the assay is capable of quantifying theamount or concentration of a nucleic acid ligand present in the testsample.

QPCR is a technique based on the polymerase chain reaction, and is usedto amplify and simultaneously quantify a targeted nucleic acid molecule.QPCR allows for both detection and quantification (as absolute number ofcopies or relative amount when normalized to DNA input or additionalnormalizing genes) of a specific sequence in a DNA sample. The procedurefollows the general principle of PCR, with the additional feature thatthe amplified DNA is quantified as it accumulates in the reaction inreal time after each amplification cycle. QPCR is described, forexample, in Kurnit et al. (U.S. Pat. No. 6,033,854), Wang et al. (U.S.Pat. Nos. 5,567,583 and 5,348,853), Ma et al. (The Journal of AmericanScience, 2(3), (2006)), Heid et al. (Genome Research 986-994, (1996)),Sambrook and Russell (Quantitative PCR, Cold Spring Harbor Protocols,(2006)), and Higuchi (U.S. Pat. Nos. 6,171,785 and 5,994,056). Thecontents of these are incorporated by reference herein in theirentirety.

Two common methods of quantification are: (1) use of fluorescent dyesthat intercalate with double-stranded DNA, and (2) modified DNAoligonucleotide probes that fluoresce when hybridized with acomplementary DNA.

In the first method, a DNA-binding dye binds to all double-stranded(ds)DNA in PCR, resulting in fluorescence of the dye. An increase in DNAproduct during PCR therefore leads to an increase in fluorescenceintensity and is measured at each cycle, thus allowing DNAconcentrations to be quantified. The reaction is prepared similarly to astandard PCR reaction, with the addition of fluorescent (ds)DNA dye. Thereaction is run in a thermocycler, and after each cycle, the levels offluorescence are measured with a detector; the dye only fluoresces whenbound to the (ds)DNA (i.e., the PCR product). With reference to astandard dilution, the (ds)DNA concentration in the PCR can bedetermined. Like other real-time PCR methods, the values obtained do nothave absolute units associated with it. A comparison of a measuredDNA/RNA sample to a standard dilution gives a fraction or ratio of thesample relative to the standard, allowing relative comparisons betweendifferent tissues or experimental conditions. To ensure accuracy in thequantification, it is important to normalize expression of a target geneto a stably expressed gene. This allows for correction of possibledifferences in nucleic acid quantity or quality across samples.

The second method uses sequence-specific RNA or DNA-based probes toquantify only the DNA containing the probe sequence; therefore, use ofthe reporter probe significantly increases specificity, and allows forquantification even in the presence of some non-specific DNAamplification. This allows for multiplexing, i.e., assaying for severalgenes in the same reaction by using specific probes with differentlycolored labels, provided that all genes are amplified with similarefficiency.

This method is commonly carried out with a DNA-based probe with afluorescent reporter (e.g. 6-carboxyfluorescein) at one end and aquencher (e.g., 6-carboxy-tetramethylrhodamine) of fluorescence at theopposite end of the probe. The close proximity of the reporter to thequencher prevents detection of its fluorescence. Breakdown of the probeby the 5′ to 3′ exonuclease activity of a polymerase (e.g., Taqpolymerase) breaks the reporter-quencher proximity and thus allowsunquenched emission of fluorescence, which can be detected. An increasein the product targeted by the reporter probe at each PCR cycle resultsin a proportional increase in fluorescence due to breakdown of the probeand release of the reporter. The reaction is prepared similarly to astandard PCR reaction, and the reporter probe is added. As the reactioncommences, during the annealing stage of the PCR, both probe and primersanneal to the DNA target. Polymerization of a new DNA strand isinitiated from the primers, and once the polymerase reaches the probe,its 5′-3′-exonuclease degrades the probe, physically separating thefluorescent reporter from the quencher, resulting in an increase influorescence. Fluorescence is detected and measured in a real-time PCRthermocycler, and geometric increase of fluorescence corresponding toexponential increase of the product is used to determine the thresholdcycle in each reaction.

In certain embodiments, the QPCR reaction uses fluorescent Taqman™methodology and an instrument capable of measuring fluorescence in realtime (e.g., ABI Prism 7700 Sequence Detector; see also PE Biosystems,Foster City, Calif.; see also Gelfand et al., (U.S. Pat. No. 5,210,015),the contents of which is hereby incorporated by reference in itsentirety). The Taqman™ reaction uses a hybridization probe labeled withtwo different fluorescent dyes. One dye is a reporter dye(6-carboxyfluorescein), the other is a quenching dye(6-carboxy-tetramethylrhodamine). When the probe is intact, fluorescentenergy transfer occurs and the reporter dye fluorescent emission isabsorbed by the quenching dye. During the extension phase of the PCRcycle, the fluorescent hybridization probe is cleaved by the 5′-3′nucleolytic activity of the DNA polymerase. On cleavage of the probe,the reporter dye emission is no longer transferred efficiently to thequenching dye, resulting in an increase of the reporter dye fluorescentemission spectra.

The nucleic acid ligand of the present invention is quantified byperforming QPCR and determining, either directly or indirectly, theamount or concentration of nucleic acid ligand that had bound to itsprobe in the test sample. The amount or concentration of the bound probein the test sample is generally directly proportional to the amount orconcentration of the nucleic acid ligand quantified by using QPCR. Seefor example Schneider et al., U.S. Patent Application Publication Number2009/0042206, Dodge et al., U.S. Pat. No. 6,927,024, Gold et al., U.S.Pat. Nos. 6,569,620, 6,716,580, and 7,629,151, Cheronis et al., U.S.Pat. No. 7,074,586, and Ahn et al., U.S. Pat. No. 7,642,056, thecontents of each of which are herein incorporated by reference in theirentirety.

Detecting the presence of the aptamer in the analyzed sample directlycorrelates to the presence of the target protein in that sample. In someembodiments of the invention, the amount of aptamer present in thesample correlates to the signal intensity following the conduction ofthe PCR-based methods. The signal intensity of PCR depends upon thenumber of PCR cycles performed and/or the starting concentration of theaptamer. Since the sequence of the target protein is known to generatethe aptamer, detection of that specific aptamer correlates to thepresence of the target protein. Similarly, detection of the amplifiedtarget nucleic acid indicates the presence of the target nucleic acid inthe sample analyzed.

In one embodiment of the invention, during amplification of the aptameror target nucleic acid using standard PCR methods, one method fordetection and quantification of amplified aptamer or target nucleic acidresults from the presence of a fluorogenic probe. In one embodiment ofthe invention, the probe, which is specific for the aptamer, has a6-carboxyfluorescein (FAM) moiety covalently bound to the 5-′end and a6-carboxytetramethylrhodamine (TAMRA) or other fluorescent-quenching dye(easily prepared using standard automated DNA synthesis) present on the3′-end, along with a 3′-phosphate to prevent elongation. The probe isadded with 5′-nuclease to the PCR assays, such that 5′-nuclease cleavageof the probe-aptamer duplex results in release of the 5′-bound FAMmoiety from the oligonucleotide probe. As amplification continues andmore aptamer is replicated by the PCR or RT-PCR enzymes, more FAM isreleased per cycle and so intensity of fluorescence signal per cycleincreases. The relative increase in FAM emission is monitored during PCRor RT-PCR amplification using an analytical thermal cycler, or acombined thermal cycler/laser/detector/software system such as an ABI7700 Sequence Detector (Applied Biosystems, Foster City, Calif.). TheABI instrument has the advantage of allowing analysis and display ofquantification in less than 60s upon termination of the amplificationreactions. Both detection systems employ an internal control or standardwherein a second aptamer sequence utilizing the same primers foramplification but having a different sequence and thus different probe,is amplified, monitored and quantitated simultaneously as that for thedesired target molecule. See for example, “A Novel Method for Real TimeQuantitative RT-PCR,” Gibson, U. et. al., 1996, Genome Res. 6:995-1001;Piatak, M. et. al., 1993, BioTechniques 14:70-81; “Comparison of the BI7700 System (TaqMan) and Competitive PCR for Quantification of IS6110DNA in Sputum During Treatment of Tuberculosis,” Desjardin, L.e. et.al., 1998, J. Clin. Microbiol. 36(7):1964-1968), the contents of whichare incorporated by reference, herein in their entirety.

In another method for detection and quantification of aptamer duringamplification, the primers used for amplification contain molecularenergy transfer (MET) moieties, specifically fluorescent resonanceenergy transfer (FRET) moieties, whereby the primers contain both adonor and an acceptor molecule. The FRET pair typically contains afluorophore donor moiety such as 5-carboxyfluorescein (FAM) or6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein (JOE), with an emissionmaximum of 525 or 546 nm, respectively, paired with an acceptor moietysuch as N′N′N′N′-tetramethyl-6-carboxyrhodamine (TAMRA),6-carboxy-X-rhodamine (ROX) or 6-carboxyrhodamine (R6G), all of whichhave excitation maximum of 514 nm. The primer may be a hairpin such thatthe 5′-end of the primer contains the FRET donor, and the 3′-end(based-paired to the 5′-end to form the stem region of the hairpin)contains the FRET acceptor, or quencher. The two moieties in the FRETpair are separated by approximately 15-25 nucleotides in length when thehairpin primer is linearized. While the primer is in the hairpinconformation, no fluorescence is detected. Thus, fluorescence by thedonor is only detected when the primer is in a linearized conformation,i.e. when it is incorporated into a double-stranded amplificationproduct. Such a method allows direct quantification of the amount ofaptamer bound to target molecule in the sample mixture, and thisquantity is then used to determine the amount of target moleculeoriginally present in the sample. See for example, Nazarenko, I. A. etal., U.S. Pat. No. 5,866,336, the contents of which is incorporated byreference in its entirety.

In another embodiment of the invention, the QPCR reaction using TaqMan™methodology selects a TaqMan™ probe based upon the sequence of theaptamer to be quantified and generally includes a 5′-end fluor, such as6-carboxyfluorescein, for example, and a 3′-end quencher, such as, forexample, a 6-carboxytetramethylfluorescein, to generate signal as theaptamer sequence is amplified using PCR. As the polymerase copies theaptamer sequence, the exonuclease activity frees the fluor from theprobe, which is annealed downstream from the PCR primers, therebygenerating signal. The signal increases as replicative product isproduced. The amount of PCR product depends upon both the number ofreplicative cycles performed as well as the starting concentration ofthe aptamer. In another embodiment, the amount or concentration of anaptamer affinity complex (or aptamer covalent complex) is determinedusing an intercalating fluorescent dye during the replicative process.The intercalating dye, such as, for example, SYBR™ green, generates alarge fluorescent signal in the presence of double-stranded DNA ascompared to the fluorescent signal generated in the presence ofsingle-stranded DNA. As the double-stranded DNA product is formed duringPCR, the signal produced by the dye increases. The magnitude of thesignal produced is dependent upon both the number of PCR cycles and thestarting concentration of the aptamer.

Nucleic acids and proteins may be obtained by methods known in the art.Generally, nucleic acids can be extracted from a biological sample by avariety of techniques such as those described by Maniatis, et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp.280-281, (1982), the contents of which is incorporated by referenceherein in its entirety. Generally, proteins can be extracted from abiological sample by a variety of techniques such as 2-Delectrophoresis, isoelectric focusing, and SDS Slab Gel Electrophoresis.See for example O'Farrell, J. Biol. Chem., 250: 4007-4021 (1975),Sambrook, J. et al., Molecular Cloning: a Laboratory Manual, 2ndEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989), Anderson et al., U.S. Pat. No. 6,391,650, Shepard, U.S. Pat. No.7,229,789, and Han et al., U.S. Pat. No. 7,488,579 the contents of eachof which is hereby incorporated by reference in its entirety.

In other embodiments, antibodies with a unique oligonucleotide tag areadded to the sample to bind a target protein and detection of theoligonucleotide tag results in detection of the protein. The targetprotein is exposed to an antibody that is coupled to an oligonucleotidetag of a known sequence. The antibody specifically binds the protein,and then PCR is used to amplify the oligonucleotide coupled to theantibody. The identity of the target protein is determined based uponthe sequence of the oligonucleotide attached to the antibody and thepresence of the oligonucleotide in the sample. In this embodiment of theinvention, different antibodies specific for the target protein areused. Each antibody is coupled to a unique oligonucleotide tag of knownsequence. Therefore, more than one target protein can be detected in asample by identifying the unique oligonucleotide tag attached to theantibody. See for example Kahvejian, U.S. Patent Application PublicationNumber 2007/0020650, hereby incorporated by reference.

In other embodiments of the invention, antibodies with a uniquenucleotide tag are added to the sample to bind the target nucleic acid.As described above, different antibodies specific for the target nucleicacid are used, therefore, more than one target nucleic acid can bedetected in a sample by identifying the unique oligonucleotide tagattached. Detection of the nucleotide tag may be done by methods knownin the art, such as PCR, QPCR, fluorescent labeling, radiolabeling,biotinylation, Sanger sequencing, sequencing by synthesis, or SingleMolecule Real Time Sequencing methods. For description of singlemolecule sequencing methods see for example, Lapidus, U.S. Pat. No.7,666,593, Quake et al., U.S. Pat. No. 7,501,245, and Lapidus et al.,U.S. Pat. Nos. 7,169,560 and 7,491,498, the contents of each of whichare herein incorporated by reference.

Antibodies for use in the present invention can be generated by methodswell known in the art. See, for example, E. Harlow and D. Lane,Antibodies, a Laboratory Model, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., (1988), the contents of which are herebyincorporated by reference in their entirety. In addition, a wide varietyof antibodies are available commercially.

The antibody can be obtained from a variety of sources, such as thoseknown to one of skill in the art, including but not limited topolyclonal antibody, monoclonal antibody, monospecific antibody,recombinantly expressed antibody, humanized antibody, plantibodies, andthe like; and can be obtained from a variety of animal species,including rabbit, mouse, goat, rat, human, horse, bovine, guinea pig,chicken, sheep, donkey, human, and the like. A wide variety ofantibodies are commercially available and a custom-made antibody can beobtained from a number of contract labs. Detailed descriptions ofantibodies, including relevant protocols, can be found in, among otherplaces, Current Protocols in Immunology, Coligan et al., eds., JohnWiley & Sons (1999, including updates through August 2003); TheElectronic Notebook; Basic Methods in Antibody Production andCharacterization, G. Howard and D. Bethel, eds., CRC Press (2000); J.Coding, Monoclonal Antibodies: Principles and Practice, 3d Ed., AcademicPress (1996); E. Harlow and D. Lane, Using Antibodies, Cold SpringHarbor Lab Press (1999); P. Shepherd and C. Dean, Monoclonal Antibodies:A Practical Approach, Oxford University Press (2000); A. Johnstone andM. Turner, Immunochemistry 1 and 2, Oxford University Press (1997); C.Borrebaeck, Antibody Engineering, 2d ed., Oxford university Press(1995); A. Johnstone and R. Thorpe, Immunochemistry in Practice,Blackwell Science, Ltd. (1996); H. Zola, Monoclonal Antibodies:Preparation and Use of Monoclonal Antibodies and Engineered AntibodyDerivatives (Basics: From Background to Bench), Springer Verlag (2000);and S. Hockfield et al., Selected Methods for Antibody and Nucleic AcidProbes, Cold Spring Harbor Lab Press (1993).

In certain embodiments, the target nucleic acid or nucleic acid ligandor both are detected using sequencing. In those embodiments, theaptamer/protein complex may be dissociated, releasing the aptamer forthe sequencing reaction. Sequencing-by-synthesis is a common techniqueused in next generation procedures and works well with the instantinvention. However, other sequencing methods can be used, includingsequence-by-ligation, sequencing-by-hybridization, gel-based techniquesand others. In general, sequencing involves hybridizing a primer to atemplate to form a template/primer duplex, contacting the duplex with apolymerase in the presence of a detectably-labeled nucleotides underconditions that permit the polymerase to add nucleotides to the primerin a template-dependent manner. Signal from the detectable label is thenused to identify the incorporated base and the steps are sequentiallyrepeated in order to determine the linear order of nucleotides in thetemplate. Exemplary detectable labels include radiolabels, florescentlabels, enzymatic labels, etc. In particular embodiments, the detectablelabel may be an optically detectable label, such as a fluorescent label.Exemplary fluorescent labels include cyanine, rhodamine, fluorescien,coumarin, BODIPY, alexa, or conjugated multi-dyes. Numerous techniquesare known for detecting sequences and some are exemplified below.However, the exact means for detecting and compiling sequence data doesnot affect the function of the invention described herein.

A sequencing technique that can be used in the methods of the providedinvention includes, for example, Helicos True Single Molecule Sequencing(tSMS) (Harris T. D. et al. (2008) Science 320:106-109). In the tSMStechnique, a DNA sample is cleaved into strands of approximately 100 to200 nucleotides, and a polyA sequence is added to the 3′ end of each DNAstrand. Each strand is labeled by the addition of a fluorescentlylabeled adenosine nucleotide. The DNA strands are then hybridized to aflow cell, which contains millions of oligo-T capture sites that areimmobilized to the flow cell surface. The templates can be at a densityof about 100 million templates/cm². The flow cell is then loaded into aninstrument, e.g., HeliScope™. sequencer, and a laser illuminates thesurface of the flow cell, revealing the position of each template. A CCDcamera can map the position of the templates on the flow cell surface.The template fluorescent label is then cleaved and washed away. Thesequencing reaction begins by introducing a DNA polymerase and afluorescently labeled nucleotide. The oligo-T nucleic acid serves as aprimer. The polymerase incorporates the labeled nucleotides to theprimer in a template directed manner. The polymerase and unincorporatednucleotides are removed. The templates that have directed incorporationof the fluorescently labeled nucleotide are detected by imaging the flowcell surface. After imaging, a cleavage step removes the fluorescentlabel, and the process is repeated with other fluorescently labelednucleotides until the desired read length is achieved. Sequenceinformation is collected with each nucleotide addition step. Furtherdescription of tSMS is shown for example in Lapidus et al. (U.S. Pat.No. 7,169,560), Lapidus et al. (U.S. patent application number2009/0191565), Quake et al. (U.S. Pat. No. 6,818,395), Harris (U.S. Pat.No. 7,282,337), Quake et al. (U.S. patent application number2002/0164629), and Braslaysky, et al., PNAS (USA), 100: 3960-3964(2003), the contents of each of these references is incorporated byreference herein in its entirety.

Another example of a DNA sequencing technique that can be used in themethods of the provided invention is 454 sequencing (Roche) (Margulies,M et al. 2005, Nature, 437, 376-380). 454 sequencing involves two steps.In the first step, DNA is sheared into fragments of approximately300-800 base pairs, and the fragments are blunt ended. Oligonucleotideadaptors are then ligated to the ends of the fragments. The adaptorsserve as primers for amplification and sequencing of the fragments. Thefragments can be attached to DNA capture beads, e.g.,streptavidin-coated beads using, e.g., Adaptor B, which contains5′-biotin tag. The fragments attached to the beads are PCR amplifiedwithin droplets of an oil-water emulsion. The result is multiple copiesof clonally amplified DNA fragments on each bead. In the second step,the beads are captured in wells (pico-liter sized). Pyrosequencing isperformed on each DNA fragment in parallel. Addition of one or morenucleotides generates a light signal that is recorded by a CCD camera ina sequencing instrument. The signal strength is proportional to thenumber of nucleotides incorporated. Pyrosequencing makes use ofpyrophosphate (PPi) which is released upon nucleotide addition. PPi isconverted to ATP by ATP sulfurylase in the presence of adenosine 5′phosphosulfate. Luciferase uses ATP to convert luciferin tooxyluciferin, and this reaction generates light that is detected andanalyzed.

Another example of a DNA sequencing technique that can be used in themethods of the provided invention is SOLiD technology (AppliedBiosystems). In SOLiD sequencing, genomic DNA is sheared into fragments,and adaptors are attached to the 5′ and 3′ ends of the fragments togenerate a fragment library. Alternatively, internal adaptors can beintroduced by ligating adaptors to the 5′ and 3′ ends of the fragments,circularizing the fragments, digesting the circularized fragment togenerate an internal adaptor, and attaching adaptors to the 5′ and 3′ends of the resulting fragments to generate a mate-paired library. Next,clonal bead populations are prepared in microreactors containing beads,primers, template, and PCR components. Following PCR, the templates aredenatured and beads are enriched to separate the beads with extendedtemplates. Templates on the selected beads are subjected to a 3′modification that permits bonding to a glass slide. The sequence can bedetermined by sequential hybridization and ligation of partially randomoligonucleotides with a central determined base (or pair of bases) thatis identified by a specific fluorophore. After a color is recorded, theligated oligonucleotide is cleaved and removed and the process is thenrepeated.

Another example of a DNA sequencing technique that can be used in themethods of the provided invention is Ion Torrent sequencing (U.S. patentapplication numbers 2009/0026082, 2009/0127589, 2010/0035252,2010/0137143, 2010/0188073, 2010/0197507, 2010/0282617, 2010/0300559),2010/0300895, 2010/0301398, and 2010/0304982), the content of each ofwhich is incorporated by reference herein in its entirety. In IonTorrent sequencing, DNA is sheared into fragments of approximately300-800 base pairs, and the fragments are blunt ended. Oligonucleotideadaptors are then ligated to the ends of the fragments. The adaptorsserve as primers for amplification and sequencing of the fragments. Thefragments can be attached to a surface and is attached at a resolutionsuch that the fragments are individually resolvable. Addition of one ormore nucleotides releases a proton (H⁺), which signal detected andrecorded in a sequencing instrument. The signal strength is proportionalto the number of nucleotides incorporated.

Another example of a sequencing technology that can be used in themethods of the provided invention is Illumina sequencing. Illuminasequencing is based on the amplification of DNA on a solid surface usingfold-back PCR and anchored primers. Genomic DNA is fragmented, andadapters are added to the 5′ and 3′ ends of the fragments. DNA fragmentsthat are attached to the surface of flow cell channels are extended andbridge amplified. The fragments become double stranded, and the doublestranded molecules are denatured. Multiple cycles of the solid-phaseamplification followed by denaturation can create several millionclusters of approximately 1,000 copies of single-stranded DNA moleculesof the same template in each channel of the flow cell. Primers, DNApolymerase and four fluorophore-labeled, reversibly terminatingnucleotides are used to perform sequential sequencing. After nucleotideincorporation, a laser is used to excite the fluorophores, and an imageis captured and the identity of the first base is recorded. The 3′terminators and fluorophores from each incorporated base are removed andthe incorporation, detection and identification steps are repeated.

Another example of a sequencing technology that can be used in themethods of the provided invention includes the single molecule,real-time (SMRT) technology of Pacific Biosciences. In SMRT, each of thefour DNA bases is attached to one of four different fluorescent dyes.These dyes are phospholinked. A single DNA polymerase is immobilizedwith a single molecule of template single stranded DNA at the bottom ofa zero-mode waveguide (ZMW). A ZMW is a confinement structure whichenables observation of incorporation of a single nucleotide by DNApolymerase against the background of fluorescent nucleotides thatrapidly diffuse in an out of the ZMW (in microseconds). It takes severalmilliseconds to incorporate a nucleotide into a growing strand. Duringthis time, the fluorescent label is excited and produces a fluorescentsignal, and the fluorescent tag is cleaved off. Detection of thecorresponding fluorescence of the dye indicates which base wasincorporated. The process is repeated.

Another example of a sequencing technique that can be used in themethods of the provided invention is nanopore sequencing (Soni G V andMeller A. (2007) Clin Chem 53: 1996-2001). A nanopore is a small hole,of the order of 1 nanometer in diameter. Immersion of a nanopore in aconducting fluid and application of a potential across it results in aslight electrical current due to conduction of ions through thenanopore. The amount of current which flows is sensitive to the size ofthe nanopore. As a DNA molecule passes through a nanopore, eachnucleotide on the DNA molecule obstructs the nanopore to a differentdegree. Thus, the change in the current passing through the nanopore asthe DNA molecule passes through the nanopore represents a reading of theDNA sequence.

Another example of a sequencing technique that can be used in themethods of the provided invention involves using a chemical-sensitivefield effect transistor (chemFET) array to sequence DNA (for example, asdescribed in US Patent Application Publication No. 20090026082). In oneexample of the technique, DNA molecules can be placed into reactionchambers, and the template molecules can be hybridized to a sequencingprimer bound to a polymerase. Incorporation of one or more triphosphatesinto a new nucleic acid strand at the 3′ end of the sequencing primercan be detected by a change in current by a chemFET. An array can havemultiple chemFET sensors. In another example, single nucleic acids canbe attached to beads, and the nucleic acids can be amplified on thebead, and the individual beads can be transferred to individual reactionchambers on a chemFET array, with each chamber having a chemFET sensor,and the nucleic acids can be sequenced.

Another example of a sequencing technique that can be used in themethods of the provided invention involves using a electron microscope(Moudrianakis E. N. and Beer M. Proc Natl Acad Sci USA. 1965 March;53:564-71). In one example of the technique, individual DNA moleculesare labeled using metallic labels that are distinguishable using anelectron microscope. These molecules are then stretched on a flatsurface and imaged using an electron microscope to measure sequences.

Sequences can be read that originate from a single molecule or thatoriginate from amplifications from a single molecule Millions ofindependent amplifications of single molecules can be performed inparallel either on a solid surface or in tiny compartments in water/oilemulsion. The DNA sample to be sequenced can be diluted and/or dispersedsufficiently to obtain one molecule in each compartment. This dilutioncan be followed by DNA amplification to generate copies of the originalDNA sequences and creating “clusters” of molecules all having the samesequence. These clusters can then be sequenced. Many millions of readscan be generated in one run. Sequence can be generated starting at the5′ end of a given strand of an amplified sequence and/or sequence can begenerated from starting from the 5′ end of the complementary sequence.In a preferred embodiment, sequence from strands is generated, i.e.paired end reads (see for example, Harris, U.S. Pat. No. 7,767,400).

Nucleotides useful in the invention include any nucleotide or nucleotideanalog, whether naturally-occurring or synthetic. For example, preferrednucleotides include phosphate esters of deoxyadenosine, deoxycytidine,deoxyguanosine, deoxythymidine, adenosine, cytidine, guanosine, anduridine. Other nucleotides useful in the invention comprise an adenine,cytosine, guanine, thymine base, a xanthine or hypoxanthine;5-bromouracil, 2-aminopurine, deoxyinosine, or methylated cytosine, suchas 5-methylcytosine, and N4-methoxydeoxycytosine. Also included arebases of polynucleotide mimetics, such as methylated nucleic acids,e.g., 2′-O-methRNA, peptide nucleic acids, modified peptide nucleicacids, locked nucleic acids and any other structural moiety that can actsubstantially like a nucleotide or base, for example, by exhibitingbase-complementarity with one or more bases that occur in DNA or RNAand/or being capable of base-complementary incorporation, and includeschain-terminating analogs. A nucleotide corresponds to a specificnucleotide species if they share base-complementarity with respect to atleast one base.

Nucleotides for nucleic acid sequencing according to the inventionpreferably include a detectable label that is directly or indirectlydetectable. Preferred labels include optically-detectable labels, suchas fluorescent labels. Examples of fluorescent labels include, but arenot limited to, 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonicacid; acridine and derivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives; eosin, eosin isothiocyanate, erythrosin and derivatives;erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives; 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives; Cy3; Cy5; Cy5.5; Cy7; IRD 700; IRD 800; LaJolta Blue; phthalo cyanine; and naphthalo cyanine. Preferredfluorescent labels are cyanine-3 and cyanine-5. Labels other thanfluorescent labels are contemplated by the invention, including otheroptically-detectable labels.

Nucleic acid polymerases generally useful in the invention include DNApolymerases, RNA polymerases, reverse transcriptases, and mutant oraltered forms of any of the foregoing. DNA polymerases and theirproperties are described in detail in, among other places, DNAReplication 2nd edition, Kornberg and Baker, W. H. Freeman, New York,N.Y. (1991). Known conventional DNA polymerases useful in the inventioninclude, but are not limited to, Pyrococcus furiosus (Pfu) DNApolymerase (Lundberg et al., 1991, Gene, 108: 1, Stratagene), Pyrococcuswoesei (Pwo) DNA polymerase (Hinnisdaels et al., 1996, Biotechniques,20:186-8, Boehringer Mannheim), Thermus thermophilus (Tth) DNApolymerase (Myers and Gelfand 1991, Biochemistry 30:7661), Bacillusstearothermophilus DNA polymerase (Stenesh and McGowan, 1977, BiochimBiophys Acta 475:32), Thermococcus litoralis (Tli) DNA polymerase (alsoreferred to as Vent™. DNA polymerase, Cariello et al., 1991,Polynucleotides Res, 19: 4193, New England Biolabs), 9.degree.Nm™. DNApolymerase (New England Biolabs), Stoffel fragment, ThermoSequenase®(Amersham Pharmacia Biotech UK), Therminator™. (New England Biolabs),Thermotoga maritima (Tma) DNA polymerase (Diaz and Sabino, 1998 Braz J.Med. Res, 31:1239), Thermus aquaticus (Taq) DNA polymerase (Chien etal., 1976, J. Bacteoriol, 127: 1550), DNA polymerase, Pyrococcuskodakaraensis KOD DNA polymerase (Takagi et al., 1997, Appl. Environ.Microbiol. 63:4504), JDF-3 DNA polymerase (from thermococcus sp. JDF-3,Patent application WO 0132887), Pyrococcus GB-D (PGB-D) DNA polymerase(also referred as Deep Vent™. DNA polymerase, Juncosa-Ginesta et al.,1994, Biotechniques, 16:820, New England Biolabs), UlTma DNA polymerase(from thermophile Thermotoga maritima; Diaz and Sabino, 1998 Braz J.Med. Res, 31:1239; PE Applied Biosystems), Tgo DNA polymerase (fromthermococcus gorgonarius, Roche Molecular Biochemicals), E. coli DNApolymerase I (Lecomte and Doubleday, 1983, Polynucleotides Res.11:7505), T7 DNA polymerase (Nordstrom et al., 1981, J. Biol. Chem.256:3112), and archaeal DP11/DP2 DNA polymerase II (Cann et al, 1998,Proc. Natl. Acad. Sci. USA 95:14250).

Both mesophilic polymerases and thermophilic polymerases arecontemplated. Thermophilic DNA polymerases include, but are not limitedto, ThermoSequenase®, 9.degree.Nm.™., Therminator™., Taq, Tne, Tma, Pfu,Tfl, Tth, Tli, Stoffel fragment, Vent™. and Deep Vent™. DNA polymerase,KOD DNA polymerase, Tgo, JDF-3, and mutants, variants and derivativesthereof. A highly-preferred form of any polymerase is a 3′exonuclease-deficient mutant.

Reverse transcriptases useful in the invention include, but are notlimited to, reverse transcriptases from HIV, HTLV-1, HTLV-II, FeLV, FIV,SIV, AMV, MMTV, MoMuLV and other retroviruses (see Levin, Cell 88:5-8(1997); Verma, Biochim Biophys Acta. 473:1-38 (1977); Wu et al., CRCCrit. Rev Biochem. 3:289-347 (1975)).

In a preferred embodiment, nucleic acid template molecules are attachedto a substrate (also referred to herein as a surface) and subjected toanalysis by single molecule sequencing as described herein. Nucleic acidtemplate molecules are attached to the surface such that thetemplate/primer duplexes are individually optically resolvable.Substrates for use in the invention can be two- or three-dimensional andcan comprise a planar surface (e.g., a glass slide) or can be shaped. Asubstrate can include glass (e.g., controlled pore glass (CPG)), quartz,plastic (such as polystyrene (low cross-linked and high cross-linkedpolystyrene), polycarbonate, polypropylene and poly(methymethacrylate)),acrylic copolymer, polyamide, silicon, metal (e.g.,alkanethiolate-derivatized gold), cellulose, nylon, latex, dextran, gelmatrix (e.g., silica gel), polyacrolein, or composites.

Suitable three-dimensional substrates include, for example, spheres,microparticles, beads, membranes, slides, plates, micromachined chips,tubes (e.g., capillary tubes), microwells, microfluidic devices,channels, filters, or any other structure suitable for anchoring anucleic acid. Substrates can include planar arrays or matrices capableof having regions that include populations of template nucleic acids orprimers. Examples include nucleoside-derivatized CPG and polystyreneslides; derivatized magnetic slides; polystyrene grafted withpolyethylene glycol, and the like.

Substrates are preferably coated to allow optimum optical processing andnucleic acid attachment. Substrates for use in the invention can also betreated to reduce background. Exemplary coatings include epoxides, andderivatized epoxides (e.g., with a binding molecule, such as anoligonucleotide or streptavidin).

Various methods can be used to anchor or immobilize the nucleic acidmolecule to the surface of the substrate. The immobilization can beachieved through direct or indirect bonding to the surface. The bondingcan be by covalent linkage. See, Joos et al., Analytical Biochemistry247:96-101, 1997; Oroskar et al., Clin. Chem. 42:1547-1555, 1996; andKhandjian, Mol. Bio. Rep. 11:107-115, 1986. A preferred attachment isdirect amine bonding of a terminal nucleotide of the template or the 5′end of the primer to an epoxide integrated on the surface. The bondingalso can be through non-covalent linkage. For example,biotin-streptavidin (Taylor et al., J. Phys. D. Appl. Phys. 24:1443,1991) and digoxigenin with anti-digoxigenin (Smith et al., Science253:1122, 1992) are common tools for anchoring nucleic acids to surfacesand parallels. Alternatively, the attachment can be achieved byanchoring a hydrophobic chain into a lipid monolayer or bilayer. Othermethods for known in the art for attaching nucleic acid molecules tosubstrates also can be used.

Any detection method can be used that is suitable for the type of labelemployed. Thus, exemplary detection methods include radioactivedetection, optical absorbance detection, e.g., UV-visible absorbancedetection, optical emission detection, e.g., fluorescence orchemiluminescence. For example, extended primers can be detected on asubstrate by scanning all or portions of each substrate simultaneouslyor serially, depending on the scanning method used. For fluorescencelabeling, selected regions on a substrate may be serially scannedone-by-one or row-by-row using a fluorescence microscope apparatus, suchas described in Fodor (U.S. Pat. No. 5,445,934) and Mathies et al. (U.S.Pat. No. 5,091,652). Devices capable of sensing fluorescence from asingle molecule include scanning tunneling microscope (siM) and theatomic force microscope (AFM). Hybridization patterns may also bescanned using a CCD camera (e.g., Model TE/CCD512SF, PrincetonInstruments, Trenton, N.J.) with suitable optics (Ploem, in Fluorescentand Luminescent Probes for Biological Activity Mason, T. G. Ed.,Academic Press, Landon, pp. 1-11 (1993), such as described in Yershov etal., Proc. Natl. Acad. Sci. 93:4913 (1996), or may be imaged by TVmonitoring. For radioactive signals, a phosphorimager device can be used(Johnston et al., Electrophoresis, 13:566, 1990; Drmanac et al.,Electrophoresis, 13:566, 1992; 1993). Other commercial suppliers ofimaging instruments include General Scanning Inc., (Watertown, Mass. onthe World Wide Web at genscan.com), Genix Technologies (Waterloo,Ontario, Canada; on the World Wide Web at confocal.com), and AppliedPrecision Inc. Such detection methods are particularly useful to achievesimultaneous scanning of multiple attached template nucleic acids.

A number of approaches can be used to detect incorporation offluorescently-labeled nucleotides into a single nucleic acid molecule.Optical setups include near-field scanning microscopy, far-fieldconfocal microscopy, wide-field epi-illumination, light scattering, darkfield microscopy, photoconversion, single and/or multiphoton excitation,spectral wavelength discrimination, fluorophor identification,evanescent wave illumination, and total internal reflection fluorescence(TIRF) microscopy. In general, certain methods involve detection oflaser-activated fluorescence using a microscope equipped with a camera.Suitable photon detection systems include, but are not limited to,photodiodes and intensified CCD cameras. For example, an intensifiedcharge couple device (ICCD) camera can be used. The use of an ICCDcamera to image individual fluorescent dye molecules in a fluid near asurface provides numerous advantages. For example, with an ICCD opticalsetup, it is possible to acquire a sequence of images (movies) offluorophores.

Some embodiments of the present invention use TIRF microscopy forimaging. TIRF microscopy uses totally internally reflected excitationlight and is well known in the art. See, e.g., the World Wide Web atnikon-instruments.jp/eng/page/products/tirf.aspx. In certainembodiments, detection is carried out using evanescent wave illuminationand total internal reflection fluorescence microscopy. An evanescentlight field can be set up at the surface, for example, to imagefluorescently-labeled nucleic acid molecules. When a laser beam istotally reflected at the interface between a liquid and a solidsubstrate (e.g., a glass), the excitation light beam penetrates only ashort distance into the liquid. The optical field does not end abruptlyat the reflective interface, but its intensity falls off exponentiallywith distance. This surface electromagnetic field, called the“evanescent wave”, can selectively excite fluorescent molecules in theliquid near the interface. The thin evanescent optical field at theinterface provides low background and facilitates the detection ofsingle molecules with high signal-to-noise ratio at visible wavelengths.

The evanescent field also can image fluorescently-labeled nucleotidesupon their incorporation into the attached template/primer complex inthe presence of a polymerase. Total internal reflectance fluorescencemicroscopy is then used to visualize the attached template/primer duplexand/or the incorporated nucleotides with single molecule resolution.

Some embodiments of the invention use non-optical detection methods suchas, for example, detection using nanopores (e.g., protein or solidstate) through which molecules are individually passed so as to allowidentification of the molecules by noting characteristics or changes invarious properties or effects such as capacitance or blockage currentflow (see, for example, Stoddart et al, Proc. Nat. Acad. Sci., 106:7702,2009; Purnell and Schmidt, ACS Nano, 3:2533, 2009; Branton et al, NatureBiotechnology, 26:1146, 2008; Polonsky et al, U.S. Application2008/0187915; Mitchell & Howorka, Angew. Chem. Int. Ed. 47:5565, 2008;Borsenberger et al, J. Am. Chem. Soc., 131, 7530, 2009); or othersuitable non-optical detection methods.

Alignment and/or compilation of sequence results obtained from the imagestacks produced as generally described above utilizes look-up tablesthat take into account possible sequences changes (due, e.g., to errors,mutations, etc.). Essentially, sequencing results obtained as describedherein are compared to a look-up type table that contains all possiblereference sequences plus 1 or 2 base errors.

In some embodiments, a plurality of nucleic acid molecules beingsequenced are bound to a solid support. To immobilize the nucleic acidon a solid support, a capture sequence/universal priming site can beadded at the 3′ and/or 5′ end of the template. The nucleic acids may bebound to the solid support by hybridizing the capture sequence to acomplementary sequence covalently attached to the solid support. Thecapture sequence (also referred to as a universal capture sequence) is anucleic acid sequence complimentary to a sequence attached to a solidsupport that may dually serve as a universal primer. In someembodiments, the capture sequence is polyN_(n), wherein N is U, A, T, G,or C, e g., 20-70, 40-60, e.g., about 50. For example, the capturesequence could be polyT₄₀₋₅₀ or its complement. As an alternative to acapture sequence, a member of a coupling pair (such as, e.g.,antibody/antigen, receptor/ligand, or the avidin-biotin pair asdescribed in, e.g., U.S. Patent Application No. 2006/0252077) may belinked to each fragment to be captured on a surface coated with arespective second member of that coupling pair.

In some embodiments, a barcode sequence is attached to the nucleic acid,the aptamer, or both. See for example, Steinman et al. (PCT internalapplication number PCT/US09/64001), the content of which is incorporatedby reference herein in its entirety.

Methods of the invention can be used to detect biomarkers, such as thosedescribed above. Examples of preferred biomarkers include FGFR3, K-ras,K-ras2, APC, DCC, TP53, PRC1, NUSAPI1, CAPZ, PFKP, EVER1, FLT1, ESPL1,AKAP2, CDC45L, RAMP, SYNGR2, NDRG1, ZNF533, and hypermethylated nucleicacid.

In certain embodiments, the disease is a cancer. Biomarkers that areassociated with cancer are known in the art. Methods of the inventioncan look for nucleic acid mutations or quantify levels of cancerassociated biomarkers to thereby diagnose a cancer. Biomarkersassociated with development of breast cancer are shown in Erlander etal. (U.S. Pat. No. 7,504,214), Dai et al. (U.S. Pat. Nos. 7,514,209 and7,171,311), Baker et al. (U.S. Pat. No. 7,056,674 and U.S. Pat. No.7,081,340), Erlander et al. (US 2009/0092973). The contents of thepatent application and each of these patents are incorporated byreference herein in their entirety. Biomarkers associated withdevelopment of cervical cancer are shown in Patel (U.S. Pat. No.7,300,765), Pardee et al. (U.S. Pat. No. 7,153,700), Kim (U.S. Pat. No.6,905,844), Roberts et al. (U.S. Pat. No. 6,316,208), Schlegel (US2008/0113340), Kwok et al. (US 2008/0044828), Fisher et al. (US2005/0260566), Sastry et al. (US 2005/0048467), Lai (US 2008/0311570)and Van Der Zee et al. (US 2009/0023137). Biomarkers associated withdevelopment of vaginal cancer are shown in Giordano (U.S. Pat. No.5,840,506), Kruk (US 2008/0009005), Hellman et al. (Br J Cancer.100(8):1303-1314, 2009). Biomarkers associated with development of braincancers (e.g., glioma, cerebellum, medulloblastoma, astrocytoma,ependymoma, glioblastoma) are shown in D'Andrea (US 2009/0081237),Murphy et al. (US 2006/0269558), Gibson et al. (US 2006/0281089), andZetter et al. (US 2006/0160762). Biomarkers associated with developmentof renal cancer are shown in Patel (U.S. Pat. No. 7,300,765), Soyupak etal. (U.S. Pat. No. 7,482,129), Sahin et al. (U.S. Pat. No. 7,527,933),Price et al. (U.S. Pat. No. 7,229,770), Raitano (U.S. Pat. No.7,507,541), and Becker et al. (US 2007/0292869). Biomarkers associatedwith development of hepatic cancers (e.g., hepatocellular carcinoma) areshown in Home et al. (U.S. Pat. No. 6,974,667), Yuan et al. (U.S. Pat.No. 6,897,018), Hanausek-Walaszek et al. (U.S. Pat. No. 5,310,653), andLiew et al. (US 2005/0152908). Biomarkers associated with development ofgastric, gastrointestinal, and/or esophageal cancers are shown in Changet al. (U.S. Pat. No. 7,507,532), Bae et al. (U.S. Pat. No. 7,368,255),Muramatsu et al. (U.S. Pat. No. 7,090,983), Sahin et al. (U.S. Pat. No.7,527,933), Chow et al. (US 2008/0138806), Waldman et al. (US2005/0100895), Goldenring (US 2008/0057514), An et al. (US2007/0259368), Guilford et al. (US 2007/0184439), Wirtz et al. (US2004/0018525), Filella et al. (Acta Oncol. 33(7):747-751, 1994), Waldmanet al. (U.S. Pat. No. 6,767,704), and Lipkin et al. (Cancer Research,48:235-245, 1988). Biomarkers associated with development of ovariancancer are shown in Podust et al. (U.S. Pat. No. 7,510,842), Wang (U.S.Pat. No. 7,348,142), O'Brien et al. (U.S. Pat. Nos. 7,291,462,6,942,978, 6,316,213, 6,294,344, and 6,268,165), Ganetta (U.S. Pat. No.7,078,180), Malinowski et al. (US 2009/0087849), Beyer et al. (US2009/0081685), Fischer et al. (US 2009/0075307), Mansfield et al. (US2009/0004687), Livingston et al. (US 2008/0286199), Farias-Eisner et al.(US 2008/0038754), Ahmed et al. (US 2007/0053896), Giordano (U.S. Pat.No. 5,840,506), and Tchagang et al. (Mol Cancer Ther, 7:27-37, 2008).Biomarkers associated with development of head-and-neck and thyroidcancers are shown in Sidransky et al. (U.S. Pat. No. 7,378,233),Skolnick et al. (U.S. Pat. No. 5,989,815), Budiman et al. (US2009/0075265), Hasina et al. (Cancer Research, 63:555-559, 2003),Kebebew et al. (US 2008/0280302), and Ralhan (Mol Cell Proteomics,7(6):1162-1173, 2008). The contents of each of the articles, patents,and patent applications are incorporated by reference herein in theirentirety. Biomarkers associated with development of colorectal cancersare shown in Raitano et al. (U.S. Pat. No. 7,507,541), Reinhard et al.(U.S. Pat. No. 7,501,244), Waldman et al. (U.S. Pat. No. 7,479,376);Schleyer et al. (U.S. Pat. No. 7,198,899); Reed (U.S. Pat. No.7,163,801), Robbins et al. (U.S. Pat. No. 7,022,472), Mack et al. (U.S.Pat. No. 6,682,890), Tabiti et al. (U.S. Pat. No. 5,888,746), Budiman etal. (US 2009/0098542), Karl (US 2009/0075311), Arjol et al. (US2008/0286801), Lee et al. (US 2008/0206756), Mori et al. (US2008/0081333), Wang et al. (US 2008/0058432), Belacel et al. (US2008/0050723), Stedronsky et al. (US 2008/0020940), An et al. (US2006/0234254), Eveleigh et al. (US 2004/0146921), and Yeatman et al. (US2006/0195269). Biomarkers associated with development of prostate cancerare shown in Sidransky (U.S. Pat. No. 7,524,633), Platica (U.S. Pat. No.7,510,707), Salceda et al. (U.S. Pat. No. 7,432,064 and U.S. Pat. No.7,364,862), Siegler et al. (U.S. Pat. No. 7,361,474), Wang (U.S. Pat.No. 7,348,142), Ali et al. (U.S. Pat. No. 7,326,529), Price et al. (U.S.Pat. No. 7,229,770), O'Brien et al. (U.S. Pat. No. 7,291,462), Golub etal. (U.S. Pat. No. 6,949,342), Ogden et al. (U.S. Pat. No. 6,841,350),An et al. (U.S. Pat. No. 6,171,796), Bergan et al. (US 2009/0124569),Bhowmick (US 2009/0017463), Srivastava et al. (US 2008/0269157),Chinnaiyan et al. (US 2008/0222741), Thaxton et al. (US 2008/0181850),Dahary et al. (US 2008/0014590), Diamandis et al. (US 2006/0269971),Rubin et al. (US 2006/0234259), Einstein et al. (US 2006/0115821), Pariset al. (US 2006/0110759), Condon-Cardo (US 2004/0053247), and Ritchie etal. (US 2009/0127454). Biomarkers associated with development ofpancreatic cancer are shown in Sahin et al. (U.S. Pat. No. 7,527,933),Rataino et al. (U.S. Pat. No. 7,507,541), Schleyer et al. (U.S. Pat. No.7,476,506), Domon et al. (U.S. Pat. No. 7,473,531), McCaffey et al.(U.S. Pat. No. 7,358,231), Price et al. (U.S. Pat. No. 7,229,770), Chanet al. (US 2005/0095611), Mitchl et al. (US 2006/0258841), and Faca etal. (PLoS Med 5(6):e123, 2008). Biomarkers associated with developmentof lung cancer are shown in Sahin et al. (U.S. Pat. No. 7,527,933),Hutteman (U.S. Pat. No. 7,473,530), Bae et al. (U.S. Pat. No.7,368,255), Wang (U.S. Pat. No. 7,348,142), Nacht et al. (U.S. Pat. No.7,332,590), Gure et al. (U.S. Pat. No. 7,314,721), Patel (U.S. Pat. No.7,300,765), Price et al. (U.S. Pat. No. 7,229,770), O'Brien et al. (U.S.Pat. No. 7,291,462 and U.S. Pat. No. 6,316,213), Muramatsu et al. (U.S.Pat. No. 7,090,983), Carson et al. (U.S. Pat. No. 6,576,420), Giordano(U.S. Pat. No. 5,840,506), Guo (US 2009/0062144), Tsao et al. (US2008/0176236), Nakamura et al. (US 2008/0050378), Raponi et al. (US2006/0252057), Yip et al. (US 2006/0223127), Pollock et al. (US2006/0046257), Moon et al. (US 2003/0224509), and Budiman et al. (US2009/0098543). Biomarkers associated with development of skin cancer(e.g., basal cell carcinoma, squamous cell carcinoma, and melanoma) areshown in Roberts et al. (U.S. Pat. No. 6,316,208), Polsky (U.S. Pat. No.7,442,507), Price et al. (U.S. Pat. No. 7,229,770), Genetta (U.S. Pat.No. 7,078,180), Carson et al. (U.S. Pat. No. 6,576,420), Moses et al.(US 2008/0286811), Moses et al. (US 2008/0268473), Dooley et al. (US2003/0232356), Chang et al. (US 2008/0274908), Alani et al. (US2008/0118462), Wang (US 2007/0154889), and Zetter et al. (US2008/0064047). Biomarkers associated with development of multiplemyeloma are shown in Coignet (U.S. Pat. No. 7,449,303), Shaughnessy etal. (U.S. Pat. No. 7,308,364), Seshi (U.S. Pat. No. 7,049,072), andShaughnessy et al. (US 2008/0293578, US 2008/0234139, and US2008/0234138). Biomarkers associated with development of leukemia areshown in Ando et al. (U.S. Pat. No. 7,479,371), Coignet (U.S. Pat. No.7,479,370 and U.S. Pat. No. 7,449,303), Davi et al. (U.S. Pat. No.7,416,851), Chiorazzi (U.S. Pat. No. 7,316,906), Seshi (U.S. Pat. No.7,049,072), Van Baren et al. (U.S. Pat. No. 6,130,052), Taniguchi (U.S.Pat. No. 5,643,729), Insel et al. (US 2009/0131353), and Van Bockstaeleet al. (Blood Rev. 23(1):25-47, 2009). Biomarkers associated withdevelopment of lymphoma are shown in Ando et al. (U.S. Pat. No.7,479,371), Levy et al. (U.S. Pat. No. 7,332,280), and Arnold (U.S. Pat.No. 5,858,655). Biomarkers associated with development of bladder cancerare shown in Price et al. (U.S. Pat. No. 7,229,770), Orntoft (U.S. Pat.No. 6,936,417), Haak-Frendscho et al. (U.S. Pat. No. 6,008,003),Feinstein et al. (U.S. Pat. No. 6,998,232), Elting et al. (US2008/0311604), and Wewer et al. (2009/0029372). The content of each ofthe above references is incorporated by reference herein in itsentirety.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

What is claimed is:
 1. A method of detecting multiple analytes in asample, the method comprising the steps of: obtaining a samplecomprising at least one target nucleic acid and at least one targetprotein; introducing an aptamer that binds to the target protein in thesample; removing unbound aptamer; and conducting a single assay, whereinthe assay detects both the target nucleic acid and the target protein,the assay comprising: performing a sequencing reaction on the targetnucleic acid and the aptamer, thereby detecting the nucleic acid and theaptamer in the sample.
 2. The method according to claim 1, furthercomprising quantifying the target nucleic acid.
 3. The method accordingto claim 1, further comprising quantifying the target protein.
 4. Themethod according to claim 1, further comprising quantifying the targetnucleic acid and the target protein.
 5. The method according to claim 1,wherein prior to sequencing, the nucleic acid and the aptamer areattached to a substrate.
 6. The method according to claim 5, wherein thenucleic acid and the aptamer are directly attached to the substrate. 7.The method according to claim 5, wherein the nucleic acid and theaptamer are indirectly attached to the substrate.
 8. The methodaccording to claim 1, wherein prior to sequencing, the method furthercomprises amplifying the nucleic acid.
 9. The method according to claim1, wherein sequencing is sequencing by synthesis.
 10. The methodaccording to claim 9, wherein the sequencing by synthesis is singlemolecule sequencing by synthesis.
 11. The method according to claim 1,wherein sequencing comprises: hybridizing a primer each of the nucleicacid and the aptamer to form each of a nucleic acid/primer duplex and aaptamer/primer duplex; contacting each of the duplexes with a polymeraseenzyme in the presence of at least one detectably labeled nucleotideunder conditions that permit the polymerase to add nucleotides to theprimers in a template-dependent manner; detecting a signal from theincorporated labeled nucleotide; and sequentially repeating thecontacting and detecting steps at least once, wherein sequentialdetection of incorporated labeled nucleotide determines the sequence ofeach of the nucleic acid and the aptamer.
 12. The method according toclaim 11, wherein the detectably labeled nucleotide is an opticallylabeled nucleotide.
 13. The method according to claim 12, wherein theoptically labeled nucleotide is a fluorescently labeled nucleotides. 14.The method according to claim 1, wherein the target nucleic acid isassociated with a disease.
 15. The method according to claim 14, whereinthe disease is cancer.
 16. The method according to claim 14, wherein thetarget nucleic acid is selected from the group consisting of: FGFR3,K-ras, K-ras2, APC, DCC, TP53, PRC1, NUSAPI1, CAPZ, PFKP, EVER1, FLT1,ESPL1, AKAP2, CDC45L, RAMP, SYNGR2, NDRG1, ZNF533, and hypermethylatednucleic acid.
 17. The method according to claim 1, wherein the targetprotein is associated with a disease.
 18. The method according to claim17, wherein the disease is cancer.
 19. The method according to claim 18,wherein the target protein is selected from the group consisting of:MMP-2, MMP-9, MMP9/NGAL complex, MMP/TIMP complex, MMP/TIMP1 complex,ADAMTS-7, ADAM-12, thymosin 15, thymosin 16, profilin, prohibitin,ubiquitin, tropomyosin, Cyr61, cystatin B, and chaperonin
 10. 20. Amethod for diagnosing a disease, the method comprising: obtaining asample comprising at least one target nucleic acid and at least onetarget protein, each of which is suspected of being associated with adisease; introducing an aptamer that binds to the target protein in thesample; removing unbound aptamer; conducting a single assay, wherein theassay detects both the target nucleic acid and the target protein, theassay comprising: performing a sequencing reaction on the target nucleicacid and the aptamer; and diagnosing the disease based on results of theassay.